The present invention relates to a process for the identification and isolation of nucleic acid molecules capable of distinguishing the isoforms PrPc and PrPSc of prion proteins as well as to the nucleic acid molecules obtainable by this process. Furthermore, the invention relates to pharmaceutical and diagnostic compositions comprising said nucleic acid molecules.
Proteinaceous infectious particles called prions are thought to be the causative agent of transmissible spongiform encephalopathies (TSEs) such as Scrapie of sheep, bovine spongiform encephalopathy (BSE) of calf, transmissible minc encephalopathy (TME) of mink as well as Kuru, Gerstmann-Strxc3xa4ussler-Scheinker syndrome (GSS), Creutzfeldt-Jakob-Disease (CJD) and fatal familial insomnia (FFI) in the case of humans (Prusiner, 1982). The main component of prions associated in amyloid-like rods (Prusiner et al., 1983; 1984) or scrapie associated fibrils (SAF; Hope et al., 1986) was found to be the prion protein PrP27-30 (Prusiner et al., 1981; Prusiner et al., 1983), an N-terminal truncated, highly protease resistant version of the prion protein PrPSc (Oesch et al., 1994), which is also found to a minor extend in prion preparations (Prusiner et al. 1983). PrP27-30, which is devoid of 67 amino acids at the aminoterminal end, results from PrPSc by proteinase K digestions (Prusiner et al., 1984; Stahl et al., 1993) or by lysosomal protease digestion (Caughey et al., 1991). The distribution of PrPSc and PrP27-30 in prion preparations varies dependent from the absence or presence of proteases.
No specific nucleic acid could be detected so far in prion preparations (Kellings et al., 1992) suggesting that the prion is infectious and can replicate in the absence of any nucleic acid (Prusiner, 1982). According to the protein-only hypothesis (Prusiner, 1982) exogenous PrPSc/PrP27-30 could convert the ubiquitous cellular isoform PrPc to PrPSc/PrP27-30. It is assumed that chaperons may be involved in this process (Edenhofer et al., 1996). PrPSc/PrP27-30 could appear as a monomer (Prusiner. 1982) or as a nucleation or crystal seed consisting of a PrPSc/PrP27-30 oligomer (Lansbury and Caughey. 1995). PrPc differs from PrP27-30 only with respect to its secondary structure: the xcex1-helical and xcex2-sheet contents of PrPc are 42% and 3%, respectively (Pan et al., 1993). In contrast. the xcex1-helical and xcex2-sheet contents of PrP27-30 were proven to be 21% and 54%. respectively (Pan et al., 1993). These results indicate that the conversion of PrPc to PrPSc/PrP27-30 is most likely concomitant with extreme alterations in the secondary structure of the prion protein. Although a series of experiments employing knock-out mice which no longer express PrPc suggest that cellular prion proteins could play a crucial role in a number of cellular processes (Collinge et al., 1994; Sakaguchi et al., 1996: Tobler et al., 1996), a precise physiological role of PrPc remains speculative. It is, however, proven that PrPc is necessary for the development of transmissible spongiform encephalopathies (Bxc3xceler et al., 1993; Brandner et al., 1996).
Translation of the mRNA from Scrapie infected Syrian golden hamster has led to a 254 amino acid protein including a 22 amino acid signal peptide at the NH2-terminus and a 23 amino acid signal sequence at the carboxy terminus (Oesch et al., 1985; Basler et al., 1986). The mature protein PrPc as well as the Scrapie isoform PrPSc contain amino acids 23 to 231. Only PrPSc can be processed to the proteinase K resistant isoform PrP27-30 (amino acids 90-231) consisting of 142 amino acids (Prusiner et al, 1984).
This property has been used to design a diagnostic assay for diseases in connection with prion proteins in which a probe is treated with proteinase K in order to degrade all PrPc and then reacted with an antibody directed against prion proteins (Groschup et al., 1994). However, this assay has the disadvantage that sensitivity might be hampered by the fact that the proteinase K digestion of PrPc is not complete, thereby leading to false positive results. Furthermore, the additional step of proteinase K digestion is time consuming. In order to be able to directly assay for the presence or absence of PrPc and/or PrPSc one would need antibodies which could distinguish between these two isoforms.
However, so far attempts to provide antibodies that can distinguish between the cellular isoform PrPc and the isoforms PrPSc, as well as the truncated version PrP27-30, have failed (Groschup et al.. 1994 and ref. therein). Thus, up to now it was not possible to distinguish the isoforms PrPc and PrPSc of prion proteins by immunological or other means which would be the prerequisite for a simple and reliable method of diagnosing a transmissible spongiform encephalopathy.
Therefore, the technical problem underlying the present invention is to provide a process for the identification and isolation of molecules which are capable of distinguishing between the isoforms PrPc and PrPSc or PrP27-30 of prion proteins and which are useful tools for diagnosis and therapy of transmissible spongiform encephalopathies.
The solution to said technical problem is achieved by the provision of the embodiments characterized by the patent claims.
Thus, the present invention relates to a process for the identification and isolation of nucleic acid molecules which are capable of distinguishing between the isoforms PrPc and PrPSc or PrP27-30 of prion proteins associated with transmissible spongiform encephalopathies comprising the steps of
(i) incubating a prion protein isoform or peptide fragment or derivative of this prion protein isoform with a pool of nucleic acid molecules comprising different sequences;
(ii) selecting and isolating those nucleic acid molecules which are capable of binding to said prion protein isoform or fragment or derivative thereof;
(iii) optionally, amplifying the isolated nucleic acid molecules and repeating steps (i) and (ii); and
(iv) determining the binding specificity of the isolated nucleic acid molecules for the PrPc and PrPSc or PrP27-30 isoforms of prion proteins.
The process according to the invention is based on a method called xe2x80x9cin vitro selectionxe2x80x9d. This method allows for the identification of nucleic acid molecules (RNA, modified RNA. ssDNA or dsDNA) which bind with high affinity to a defined molecular target from a large randomized population of nucleic acid molecules (Tuerk and Gold, 1990; Famulok and Szostak, 1992). Using this method it has been possible to isolate nucleic acids specifically recognizing a variety of protein targets including HIV-1 reverse transcriptase (Tuerk et al., 1992), HIV-1 Integrase (Allen et al., 1995), human xcex1-thrombin (Kubik et al., 1994) and Drosphila sex-lethal protein (Sakashita and Sakamoto, 1994). However, up to now it has not been possible to provide by this method nucleic acid molecules being capable of distinguishing the two isoforms of prion proteins, PrPc and PrPSc. In the scope of the present invention the term PrPc comprises the cellular isoform of the prion protein as well as fragments and derivatives thereof irrespective of the source organism. The term PrPSc comprises the isoform of the prion protein associated with various transmissible spongiform encephalopathies. This term also comprises fragments of this prion protein isoform such as the truncated version of the isoform PrPSc, the prion protein PrP27-30, which is the main component of prions. In particular, this term also includes PrPSc proteins of the various Scrapie strains including those adapted to hamster, mouse or other vertebrates. Also included are derivatives of the prion protein isoform PrPSc.
The term derivatives includes chemically modified versions of the prion protein isoforms PrPc and PrPSc as well as mutants of these proteins, namely proteins which differ from the naturally occurring prion protein isoforms at one or more positions in the amino acid sequence, as well as proteins that show deletions or insertions in comparison to the naturally occurring prion protein isoforms. Such mutants can be produced by recombinant DNA technology or can be naturally occurring mutants. The term derivatives also embraces proteins which contain modified amino acids or which are modified by glycosylation, phosphorylation and the like.
According to the invention it is possible to use as nucleic acid molecules single or double stranded nucleic acid molecules, such as RNA, modified RNA, single stranded DNA or double stranded DNA.
A pool of nucleic acid molecules, which constitutes the starting material from which nucleic acid molecules are selected which specifically bind to one of the isoforms of the prion protein is defined as a mixture of nucleic acid molecules of different sequences. This pool can be any mixture of nucleic acid molecules, preferably a pool of randomized molecules. Preferably the nuclelic acid molecules of the pool are chemically synthesized or produced by in vitro transcription.
In the case of RNA molecules the RNA pool which is screened for molecules specifically binding to one of the isoforms of a prion protein is preferably the RNA pool M111.1 described in Famulok (1994). This pool consists of RNA molecules of 111 nucleotides randomized at 74 positions and results from the transcription of corresponding DNA sequences. The pool M111.1 contains RNA molecules with approximately 1xc3x971015 different sequences.
The process according to the invention can be used to identify and isolate nucleic acid molecules which can distinguish between the two isoforms of prion proteins, PrPc and PrPSc, associated with a transmissible spongiform encephalopathy such as Scrapie of sheep, bovine spongiform encephalopathy (BSE) of calf, transmissible mink encephalopathy (TME) of mink, Kuru, Gerstmann-Strxc3xa4ussler-Scheinker Syndrome (GSS), fatal familial insomnia (FFI), Creutzfeldt-Jakob Disease (CJD) in the case of humans, chronic wasting disease (CWD) of mule, deer and elk or feline spongiform encephalopathy (FSE) of cats. Transmissible spongiform encephalopathies are also known from nyala, gemsbok, arabian oryx, greater kudu, eland, ankole, moufflon, puma, cheetah, scimitar horned oryx, ocelot and tiger.
The step of incubating the pool of nucleic acid molecules with a prion protein can be carried out in different ways. In one preferred embodiment of the invention the protein is immobilized, for example, on a matrix such as a gel or a resin for chromatography. The immobilization can be achieved by means known to the person skilled in the art. For example, the protein can be covalently linked to a matrix or can be bound to it by a specific interaction between a group present on the matrix and a domain of the protein specifically recognizing this group. Such a domain can be fused to a prion protein by recombinant DNA technology as will be discussed below.
If the prion protein is immobilized nucleic acid molecules which do not bind to the prion protein can be removed after incubation by washing with an appropriate buffer Subsequently the nucleic acid molecules binding to the prion protein can be eluted from the immobilized protein, for example by 8M urea, and further purified, for example, by phenol extraction and precipitation.
In another preferred embodiment the prion protein is in solution. In this case the nucleic acid molecules binding to the prion protein can be isolated, for example, by carrying out a gel retardation assay and isolating the protein/nucleic acid complex. Subsequently the nucleic acid molecules can be isolated from the complex and further purified by known methods.
According to the invention it is possible to amplify the nucleic acid molecules obtained by steps (i) and (ii), for example by in vitro transcription, reverse transcription or polymerase chain reaction or a combination of these techniques, and to repeat steps (i) and (ii). This leads to a further selection and amplification of nucleic acid molecules which bind specifically to the used prion protein. If several cycles of steps (i) to (iii) of the process are performed, it is possible to use in one or more cycles an immobilized protein and in one or more cycles a protein in solution. A cycle in which a protein in solution is used permits the elimination of nucleic acid molecules binding to the matrix on which the immobilized protein is fixed.
The prion protein used in the process can be any of the known prion protein isoforms or a fragment or derivative of such a protein.
In a preferred embodiment the prion protein is the isoform PrPSc present in the prion. In a specifically preferred embodiment the N-terminally truncated version of PrPSSc, PrP27-30, is used. In this context PrPSc and PrP 27-30 refer to any of these isoforms which can be found in an organism affected with a transmissible spongiform encephalopathy.
In a further preferred embodiment the prion protein used in the process is the cellular isoform PrPc, most preferably the processed form PrPc23-231 which comprises amino acids 23 to 231 of PrPc.
In another preferred embodiment the prion protein used in the process is a recombinant protein. This means that the protein is produced by recombinant DNA technology, namely by expression from a cloned DNA sequence.
More preferably, the prion protein is part of a fusion protein. Such a fusion protein can comprise beside the prion protein a protein or protein domain which confers to the fusion protein a specific binding capacity. For example, such a domain may be an oligohistidine (Le-Grice et al., 1990), Calmoduline binding peptide (CBP) (Carr et al., 1991), S-peptide (ribonuclease A) (Kim and Raines, 1993), FLAG (Kawase et al., 1995), green-fluorescent protein (GFP) (Hampton et al., 1996), BTag (Wang et al., 1996), or maltose-binding protein (MBP) (Aitken et al., 1994, Richards and Wyckoff, 1971). Proteins comprising such a domain can be immobilized for example, on IMAC-Ni2+, Calmodulin, S-protein 104 aa (Kim and Raines, 1993), anti-FLAG-antibodies, anti-GFP-antibodies, BTag-antibodies or maltose. Elution can then be achieved by a method well-known in the art. In a preferred embodiment the prion protein is fused to glutathione-S-transferase. Such a fusion protein possesses a high affinity for glutathione and can thus be immobilized on a matrix comprising glutathione, such as glutathione-sepharose.
In the last step of the process according to the invention the isolated nucleic acid molecules are tested for their binding to the different isoforms, PrPc and PrPSc, of a prion protein. Those nucleic acid molecules are selected which specifically bind to only one of the isoforms.
Thus, the process according to the invention allows the identification and isolation of nucleic acid molecules which specifically bind to one of the isoforms of a prion protein or a fragment or derivative thereof and thereby allow the distinguishing of the different isoforms. These nucleic acid molecules therefore show an unexpected high specificity, which is even higher than the specificity of poly- or monoclonal antibodies which cannot distinguish between the isoforms of prion proteins. The process of the invention has been successfully carried out to isolate RNA molecules which can distinguish between the isoforms PrPc23-231 and PrP27-30 from Syrian Golden Hamster. In this case the isoforms were recombinant proteins fused to glutathicne-s-transferase (GST::PrPc23-231 and GST::rPrP27-30). The recombinant rPrP27-30 protein is identical in sequence to the natural PrP27-30 protein but reveals in contrast to the natural isoform proteinase K sensitivity.
Furthermore, the present invention relates to nucleic acid molecules obtainable by a process according to the invention, namely to RNA, single stranded DNA or double stranded DNA molecules which bind to one of the isoforms of a prion protein. These include nucleic acid molecules which specifically bind to the cellular isoform PrPc, namely to the processed form PrPc23-231, or specifically to the isoform PrPSc, namely to the truncated version PrP27-30, or specifically to derivatives of these proteins.
In a preferred embodiment the nucleic acid molecules of the invention comprise four stretches of three consecutive guanosine residues separated by single stranded regions between four and seven nucleotides long. More preferably, the nucleic acid molecules comprise a nucleotide sequence as depicted in SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.
In another preferred embodiment, the region comprising the four guanosine stretches is flanked by two variable regions of predominantly Watson-Crick covariation. In particular, the nucleic acid molecules preferably comprise a nucleotide sequence as depicted in any one of SEQ ID NO: 1 to 13 and more preferably a nucleotide sequence as depicted in SEQ ID NO: 18.
In a preferred embodiment the nucleic acid molecules according to the invention are further modified at one or more positions in order to increase their stability and/or to alter their biochemical and/or biophysical properties.
The present invention also relates to pharmaceutical compositions comprising nucleic acid molecules according to the invention. Such compositions can optionally comprise pharmaceutically acceptable carriers.
These compositions may be useful for the therapy of transmissible spongiform encephalopathies such as those listed above. It may be possible, for example, to suppress the conversion of the isoform PrPc into the prion associated isoform PrPSc by applying nucleic acid molecules which specifically bind to PrPc.
Furthermore, the present invention relates to diagnostic compositions comprising nucleic acid molecules according to the invention. Such compositions may contain additives commonly used for diagnostic purposes. The nucleic acid molecules and the diagnostic compositions according to the invention can be used in methods for the diagnosis of transmissible spongiform encephalopathies. Such a method comprises, for example, the incubation of a sample taken from a body with at least one kind of nucleic acid molecules according to the invention and the subsequent determination of the interaction of the nucleic acid molecules with the isoforms PrPc and PrPSc of a prion protein.
Since during the occurrence of a transmissible spongiform encephalopathy the amount of the isoform PrPSc increases and the total amount of the cellular isoform PrPc decreases, it is in principle possible to use for diagnosis nucleic acid molecules which bind to one or the other of the two isoforms.
On the one hand, it is possible to use at least one kind of nucleic acid molecule according to the invention in order to quantitatively determine the amount of at least one isoform of a prion protein in a sample.
On the other hand, it is possible to use nucleic acid molecules which specifically bind the PrPc isoform in combination with nucleic acid molecules which specifically bind the PrPSc isoform in order to determine the absolute and/or relative amount of the isoforms in a sample.
In a preferred embodiment the sample may be obtained from various organs, perferably from tissue, for example, from brain, tonsils, ileum, cortex, dura mater, Purkinje cells, lymphnodes, nerve cells, spleen, muscle cells, placenta, pancreas, eyes, backbone marrow or peyer""sche plaques, for example in the form of thin sections. Alternatively the sample may be obtained from a body fluid, preferably from blood, cerebrospinal fluid, milk or semen.
In the case that brain is used as a sample, diagnosis is in most cases performed post mortem. Exceptionally, brain biopsies can be performed on the alive organism. The brain can originate from any organism that might be afflicted with a transmissible spongiform encephalopathy, such as sheep, calf, mice, cats, hamster, mule, deer, elk or humans or from other organisms which may be afflicted by a TSE as mentioned above. The brain should originate from organisms which are PrP0/0 (knock-out), PrPSc (infected) and PrPc (wild-type) or of unknown PrP-status.
In the case that blood, milk, cerebrospinal fluid, semen or tissue from other organs as mentioned above is used as a sample, diagnosis is possible for living individuals.
Furthermore, the nucleic acid molecules according to the invention can be used to identify three dimensional structures which are necessary for the specific binding of a prion protein isoform. With the help of this information other chemical compounds can be isolated or synthesized which can specifically bind prion protein isoforms. Thus, the present invention also relates to chemical compounds other than nucleic acid molecules which are based on the information derived from a three dimensional structure of a nucleic acid molecule according to the invention, selected from the group consisting of inorganic or organic compounds, preferably sugars, amino acids, proteins or carbohydrates.